Cathepsin B-independent abrogation of cell death by CA-074-OMe upstream of lysosomal breakdown.
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Amino Acid Chloromethyl Ketones↗
Biomedical subjects
R Mihalik
Publications and source records attributed to R Mihalik.
Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells.
In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.
Amino Acid Chloromethyl Ketones↗
The somatostatin analogue peptide TT-232 induces apoptosis and chromosome breakage in cultured human lymphocytes.
Somatostatin receptors are supposed to be important in the regulation of apoptosis. In this study, we measured apoptosis occurring spontaneously, or induced by the synthetic somatostatin analogue, the peptide TT-232. We examined isolated human peripheral blood lymphocytes (PBL) from 32 nurses exposed bedside to cytostatic drugs, 12 chronic lymphoid leukaemia (CLL) patients prior to treatment, and 19 unexposed, healthy donors without anamnestic occupational exposure to genotoxic agents. Cells were stimulated by phytohaemagglutinin-P (PHA) and cultured for 69 h with or without 15 microg/ml TT-232, respectively. Cell kinetic parameters and apoptosis were determined by flow cytometry after staining with FITC-labeled anti-BrdU and propidium iodide (PI) and the results on spontaneous and peptide-induced apoptosis were compared with the obtained chromosome aberration frequencies (CA). The peptide TT-232 unexpectedly induced chromosome breakage in addition to apoptosis. The mean spontaneous apoptotic fractions were 6.65+/-0.89%, 6.46+/-0. 53%, and 3.07+/-0.57%, and the mean CA yields in the samples without TT-232 were 1.74+/-0.46%, 2.44+/-0.40%, and 4.50+/-1.05%, for healthy subjects, nurses, and CLL patients, respectively. A total of 15 microg/ml TT-232 treatment in healthy subjects increased the mean CA frequency (10.38+/-1.57%), as well as the apoptotic cell fraction (2.63+/-0.45 times higher than the corresponding untreated sample). In TT-232-treated PBLs of nurses, CA remained unchanged and the mean apoptotic cell fraction showed only a slight increase (1.24+/-0.11 times higher than the untreated). Among CLL patients, TT-232 treatment significantly increased both CA (up to 17.83+/-4.04%) and the ratio of apoptotic cells (21.78+/-11.00 times higher than the untreated). These results demonstrated significant differences in apoptosis sensitivity in controls, nurses and CLL donors, after 15 microg/ml TT-232 treatment. Data also indicate that the induced CA yields in CLL donors with high CA are in correlation with TT-232-induced apoptosis.
Antineoplastic Agents↗
Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcinoma cells.
Apoptosis is a specific mode of programmed cell death (PCD), recognized by characteristic morphological and molecular changes. Here we present evidence for a non-apoptotic type of PCD in human MCF-7 breast carcinoma cells. We used TNF-alpha and tyrphostin AG213 to induce apoptotic and non-apoptotic cell death respectively in vitro. Microscopic and immunohistochemical studies, together with DNA analysis and flow cytometric analysis of p53 and bcl-2 oncogene expression, revealed some novel characteristics of non-apoptotic cell death. We show here for the first time some of the biochemical features of an experimentally induced non-apoptotic PCD and emphasize the distinct biochemical events leading to apoptotic and non-apoptotic PCD.
Apoptosis↗
Apoptosis inhibitory effect of the isothiourea compound, tri-(2-thioureido-S-ethyl)-amine.
Sulfhydril compounds, some of them with immunomodulatory activity have also been shown to modulate the induction of apoptosis. This study was performed to assess the possible apoptosis inhibitory effect of the immunomodulatory compound tri-(2-thioureido-S-ethyl)-amine (K-1) in U-937 and HL-60 leukaemia cell lines as model systems. Treatment of U-937 and HL-60 cells with K-1 inhibited etoposide (ETO)-induced apoptosis in both cell lines in a dose-dependent manner, IC(50)=200 microg/ml. The results indicate that inhibition of ETO-induced apoptosis occurs downstream of ETO-mediated cleavable complex formation but early in, or at the level of the mitochondria events of the apoptotic pathway. Further, like other isothioureas, K-1 proved to be a potent inhibitor of nitric oxide synthase. This raises the possibility that where the activation of nitric oxide synthase is involved in apoptosis induction, K-1 might also be effective. These findings suggest that K-1 may serve as a potent inhibitor of apoptosis initiated by ETO or nitric oxide.
Animals↗
Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.
The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.
Animals↗
Interaction of cytocidal drugs and the inhibition of caspase-3 by 3-nitrosobenzamide.
The effect of 3-nitrosobenzamide (NOBA) on the etoposide, staurosporine and dexamethason induced rapid (4-6 hr), caspase-dependent apoptosis was investigated in thymocytes and lymphoma cells by flow cytometric assay of DNA fragmentation. When NOBA (ED(50) = 4 microM) was added to these cell systems, the rapid onset of apoptosis was prevented. Such apparent protection by NOBA was related to the inactivation of caspase-3, by s-nitrosylation of 1.3 mol -SH per enzyme molecule out of 7 -SH groups. Since NOBA by itself induces DNA fragmentation within 18 hr in lymphoma cells, our results indicate that at least two active cell death pathways exist with apparent dissimilar kinetics and molecular mechanisms.
Animals↗
[Cell death and tumor formation].
Proliferation and death (apoptosis) are the main cellular functions in the maintenance of the homeostasis of the organism. Disturbances in the regulation of these two basic phenomena may results in the continuous and irreversible accumulation of cells in time and space. This process which could become autonomous without any obvious biological "advantage". Both the overproduction of anti-apoptotic factors (e.g. bcl-2) or the failure of pro-apoptotic signals (e.g. p53) can contribute to tumorigenesis. Authors review the different phases of apoptosis with emphasis on the most important decision making elements in cell death, i.e. caspases and mitochondria, as well as on the interactions of the regulatory pathways. The better understanding of these coordinated and interdependent actions may help to achieve improvements in many fields of oncology.
Apoptosis↗
Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.
Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.
Animals↗
Syndecan-1 (CD138) expression in human non-Hodgkin lymphomas.
Syndecan-1, an important transmembrane heparan sulphate proteoglycan is expressed in distinct stages of differentiation of normal lymphoid cells: in pre-B cells and Ig-producing plasma cells; however, its normal function, or presence in lymphoid malignancies, is still largely unknown. The expression of syndecan-1 (CD138) was studied in 57 human non-Hodgkin lymphomas using immunocytochemistry and immunohistochemistry. Positive expression of syndecan-1 was found in the plasma cells in chronic lymphoblastic leukaemia (B-CLL) cases, in different plasmocytoid lymphomas as well as in myeloma. All normal and malignant Tcells, or CD5+ cells other than B-CLL proved to be negative. These results strongly suggest that syndecan-1 expression is a characteristic phenotypic marker for B-CLL and lymphoplasmocytoid lymphomas and could be used for diagnostic purposes.
Biomarkers, Tumor↗
Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines.
Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.
Alkynes↗
BCNU is a caspase-mediated inhibitor of drug-induced apoptosis.
BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea], a bifunctional (alkylating/carbamoylating) anticancer agent, in noncytotoxic doses (12-50 microM) inhibited drug-induced apoptosis in HT58 human lymphoma cells exposed to etoposide (ETO; 50 microM) as well as in mouse thymocytes exposed to dexamethasone (5 microg/ml) in vitro in 4-h cultures. The cytoplasmic extracts of ETO-treated HT58 cells cleaved both purified poly(ADP-ribose)polymerase and Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin fluorogenic caspase substrate, indicating the presence of active caspases, and these effects were inhibited by BCNU concentration dependently. The carbamoylating decomposite, 2-chloroethyl-isocyanate (6-25 microM), also decreased ETO-induced apoptosis in HT58 cells in vitro and their caspase 3-like activity ex vivo, whereas N-(2-chloroethyl)-N-nitrosocarbamoyl-valinamide, an alkylating and mainly intramolecularly carbamoylating nitrosourea derivative (400 microM), did not influence these phenomena. Furthermore, the activity of recombinant caspase 3 was also strongly inhibited by BCNU and 2-chloroethyl-isocyanate. These results indicate that BCNU, via its carbamoylating capacity, can inactivate cysteine protease(s) essential for ETO-induced apoptosis. This apoptosis-modulating property of BCNU, in turn, may influence the efficacy of chemotherapeutic protocols in the treatment of cancer.
Antineoplastic Agents, Alkylating↗
Apoptotic cell death induced by inhibitors of energy conservation--Bcl-2 inhibits apoptosis downstream of a fall of ATP level.
Energy charge controls intermediary metabolism and cellular regulation. Here we show that inhibition of energy conservation at the level of glucose uptake, glycolysis, citric acid cycle, and oxidative phosphorylation induces cell death, leading to fragmentation of DNA into an oligonucleosomal ladder and morphological changes typical for apoptosis. Bcl-2, the prototype of oncogenes that suppress cell death, efficiently inhibits apoptosis induced by metabolic inhibitors. Bcl-2 does not antagonize the inhibitory potential of mitochondrial inhibitors, and cannot prevent or delay the decrease of the cellular ATP level subsequent to metabolic inhibition. Thus, we propose that Bcl-2 blocks apoptosis at a point downstream of the collapse of the cellular-energy homeostasis.
Adenosine Triphosphate↗
Expression of syndecan-1 in human B cell chronic lymphocytic leukaemia.
Syndecan-1 is considered an important transmembrane proteoglycan in cell-microenvironment interactions, but its exact function in normal or in transformed B cells is still unknown. In this study, RNA was isolated from peripheral cells of chronic lymphocytic leukaemia (B-CLL) and 'normal', non-leukaemic patients, as controls. Reverse PCR showed no or very low syndecan-1 mRNA expression in controls, while in 11/13 B-CLL the circulating leukaemic cells expressed syndecan-1. Similar results were obtained for interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6). Furthermore, syndecan-1 protein was detected in the majority of circulating B-CLL cells by flow cytometry and immunocytochemistry using anti-syndecan-1 MAb. Control cells were practically negative. Further study is required to understand the biological significance of syndecan-1 on B-CLL cells.
Adult↗
Characterisation of the swine swC1 antigen.
Some biochemical and functional characteristics of the swine swC1 antigen, determined by the use of the authors' swC1-specific monoclonal antibody (mAb) 335-2, are reported. The molecular weight of the antigen was determined by immunoprecipitation. The swC1 antigen has 41 and approx. 15 kD components under reducing conditions. It is sensitive to proteolytic enzymes such as bromelain or trypsin, but not to papain. Phosphatidylinositol-specific phospholipase C treatment diminished the expression of swC1 on the surface of leukocytes. Cross-linking of swC1 on the cell surface did not influence the proliferation of mitogen-activated mononuclear cells and had no mitogenic activity by itself. During 48 h of mitogen activation its surface expression did not change significantly. Possible relationships of swC1 to human CD antigens are discussed in the light of the results obtained.
Animals↗
Possible involvement of protein kinase C-epsilon in phorbol ester-induced growth inhibition of human lymphoblastic cells.
Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional alpha, beta and the novel epsilon isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (o.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5-1.0 ng/ml PMA. The translocation of PKC-epsilon was observed within 39 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and and altered phosphorylation state of PKC-epsilon was seen. In contrast, the conventional alpha and beta isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the alpha and beta isoforms showed a significant down-regulation. The preferential alterations in PKC-epsilon observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca(2+)-independent epsilon isoform in mediating the initial events of the phorbol ester stimulated cellular responses.
Apoptosis↗
Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors.
The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and demecolcine lead to accumulation of cells primarily in G1/S, S, S/G2/M, G2/M, and M stages of the cell cycle, respectively. While no significant change in TNF receptors occurred in cells arrested in G1/S or S/G2 stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G1 and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response.
Cell Cycle↗
The surface phenotype of swine blood and tissue eosinophil granulocytes.
Cell surface antigens of swine eosinophil granulocytes were studied with flow cytometry and immunohistochemistry. The monoclonal antibody 335-2, specific for swine differentiation antigen swC1a, originally described to be present on swine T and myeloid cells, is able to distinguish swine eosinophils (swC1a negative) from neutrophils (swC1a positive). This monoclonal antibody (mAb) was used in two-colour fluorescence measurements in combination with anti-swine -CD2, -CD4, -CD8, -MHC class II, -LFA-1 or -swC3 mAbs. All of the blood eosinophils proved to be positive for LFA-1 and swC3, a common marker of swine monocytes, granulocytes and macrophages. However, they do not react with antibodies recognizing swine CD2, CD4, CD8 or MHC class II cell surface molecules. The reactivity pattern of tissue eosinophils with these mAbs was determined on cryostat sections of different tissues of swine. Tissue eosinophils were negative for swC1a, CD2, CD8, while all of them reacted with swC3. In contrast with blood eosinophils, 10-30% of tissue eosinophils were demonstrated to be negative for LFA-1. In some cases, a few tissue eosinophils were found to be stained weakly by antibodies to swine CD4 or MHC class II antigens.
Animals↗